Treating stroke and other diseases without inhibiting N-type calcium channels

ABSTRACT

The invention provides methods for treating stroke and compositions for use in the same. The methods employ a chimeric peptide of an active peptide and an internalization peptide. The internalization peptide is a tat variant that promotes uptake of itself and a linked active peptide into a cell without substantial binding to N-type calcium channels. Use of the tat variant allows treating of stroke free of certain side effects associated with binding to N-type calcium channels. Tat variant peptides can also be linked to other active agent for use in treating other diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a non-provisional and claims the benefit ofU.S. Ser. No. 60/904,507, filed Mar. 2, 2007, incorporated by referencein its entirety for all purposes.

BACKGROUND OF THE INVENTION

Stroke is predicted to affect more than 600,000 people in the UnitedStates a year. In a 1999 report, over 167,000 people died from strokes,with a total mortality of 278,000. In 1998, 3.6 billion was paid to justthose Medicare beneficiaries that were discharged from short-stayhospitals, not including the long term care for >1,000,000 people thatreportedly have functional limitations or difficulty with activities ofdaily living resulting from stroke (Heart and Stroke Statistical update,American Heart Association, 2002). No therapeutic has been approved toreduce brain damage resulting from stroke through the direct protectionneurons from death.

Stroke is characterized by neuronal cell death in areas of ischemia,brain hemorrhage and/or trauma. Cell death is triggered by glutamateover-excitation of neurons, leading to increased intracellular Ca²⁺ andincreased nitric oxide due to an increase in nNOS (neuronal nitric oxidesynthase) activity.

Glutamate is the main excitatory neurotransmitter in the central nervoussystem (CNS) and mediates neurotransmission across most excitatorysynapses. Three classes of glutamate-gated ion channel receptors(N-methyl-D-aspartate (NMDA),alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) andKainate) transduce the postsynaptic signal. Of these, NMDA receptors(NMDAR) are responsible for a significant portion of the excitotoxicityof glutamate. NMDA receptors are complex having an NR1 subunit and oneor more NR2 subunits (2A, 2B, 2C or 2D) (see, e.g., McDain, C. andCaner, M. (1994) Physiol. Rev. 74:723-760), and less commonly, an NR3subunit (Chatterton et al. (2002) Nature 415:793-798). The NR1 subunitshave been shown to bind glycine, whereas NR2 subunits bind glutamate.Both glycine and glutamate binding are required to open the ion channeland allow calcium entry into the cell. The four NR2 receptor subunitsappear to determine the pharmacology and properties of NMDA receptors,with further contributions from alternative splicing of the NR1 subunit(Kornau et al. (1995) Science 269:1737-40). Whereas NR1 and NR2Asubunits are ubiquitously expressed in the brain, NR2B expression isrestricted to the forebrain, NR2C to the cerebellum, and NR2D is rarecompared to the other types.

Because of the key role of NMDA receptors in the excitotoxicityresponse, they have been considered as targets for therapeutics.Compounds have been developed that target the ion channel (ketamine,phencyclidine, PCP, MK801, amantadine), the outer channel (magnesium),the glycine binding site on NR1 subunits, the glutamate binding site onNR2 subunits, and specific sites on NR2 subunits (Zinc-NR2A; Ifenprodil,Traxoprodil-NR2B). Among these, the non-selective antagonists of NMDAreceptor have been the most neuroprotective agents in animal models ofstroke. However, clinical trials with these drugs in stroke andtraumatic brain injury have so far failed, generally as a result ofsevere side effects such as hallucination and even coma.

Postsynaptic density-95 protein (PSD-95) has been reported to coupleNMDARs in pathways mediating excitotoxicity and ischemic brain damage(Aarts et al., Science 298, 846-850 (2002)). This coupling was disruptedby transducing neurons with peptides from the C-terminus of NMDAR 2Bthat bind to PSD-95 fused to a standard tat internalization peptide.This treatment attenuated downstream NMDAR signaling without inhibitingNMDAR activity, protected cultured cortical neurons from excitotoxicinsults and reduced cerebral infarction volume in rats subjected totransient focal cerebral ischemia.

BRIEF SUMMARY OF THE INVENTION

The invention provides an isolated chimeric peptide or peptidomimeticthereof, wherein the chimeric peptide comprises an active peptide thatinhibits binding of PSD-95 to an NMDA receptor and an internalizationpeptide that promotes uptake of the chimeric peptide into cells and hasreduced capacity to bind to an N-type calcium channel relative to thetat peptide YGRKKRRQRRR (SEQ ID NO:1). Optionally, the internalizationpeptide is a variant of the tat peptide. Optionally, the active peptidehas an amino acid sequence consisting of 3-25 amino acids from theC-terminus of an NMDA receptor or a PDZ domain 1 and/or 2 from a PSD-95receptor. Optionally, the active peptide has an amino acid sequencecomprising T/SXV/L (SEQ ID NO:14) and the internalization peptide has anamino acid sequence comprising XGRKKRRQRRR (SEQ ID NO:2), wherein X isan amino acid other than Y or nothing. Optionally, X is F (SEQ IDNO:135). Optionally, X is nothing (SEQ ID NO:136). Optionally theinternalization peptide consists of GRKKRRQRRRP (SEQ ID NO:3).Optionally, the chimeric peptide has an amino acid sequence consistingof GRKKRRQRRRKLSSIESDV (SEQ ID NO:4).

Optionally, the active peptide has an amino acid sequence comprising[E/D/N/Q]-[S/T]-[D/E/Q/N]-[V/L] (SEQ ID NO:5). Optionally, the activepeptide comprises an amino acid sequence selected from the groupconsisting of ESDV (SEQ ID NO:6), ESEV (SEQ ID NO:7), ETDV (SEQ IDNO:8), ETEV (SEQ ID NO:9), DTDV (SEQ ID NO:10), DTEV (SEQ ID NO:11).Optionally, the active peptide has an amino acid sequence comprisingKLSSIESDV (SEQ ID NO:12). Optionally, the active peptide has an aminoacid sequence comprising KLSSIETDV (SEQ ID NO:13). Optionally, thechimeric peptide has an amino acid sequence comprisingFGRKKRRQRRRKLSSIESDV (SEQ ID NO:19) or FGRKKRRQRRRKLSSIETDV (SEQ IDNO:16). Optionally, the chimeric peptide has an amino acid sequenceconsisting of FGRKKRRQRRRKLSSIESDV (SEQ ID NO:19) orFGRKKRRQRRRKLSSIETDV (SEQ ID NO:16). Optionally, the chimeric peptidehas a Kd greater than 10 nM for an N-type calcium channel.

The invention further provides a pharmaceutical composition comprisingan isolated chimeric peptide or peptidomimetic thereof as describedabove and a pharmaceutically acceptable carrier.

The invention further provides a method of treating the damaging effectof stroke in a patient having or at risk of stroke or other injury tothe CNS, comprising administering to the patient an effective amount ofa chimeric peptide or peptidomimetic thereof. The chimeric peptidecomprises an active peptide having an amino acid sequence comprisingT/SXV/L (SEQ ID NO:14) and a internalization peptide having an aminoacid sequence comprising XGRKKRRQRRR (SEQ ID NO:2), wherein X is anamino acid other than Y. Optionally, X is F (SEQ ID NO:135). Optionally,X is nothing (SEQ ID NO:136). Optionally, the internalization peptideconsists of GRKKRRQRRRPQ (SEQ ID NO:15). Optionally, the chimericpeptide has an amino acid sequence comprising GRKKRRQRRRKLSSIESDV (SEQID NO:4). Optionally, the active peptide has an amino acid sequencecomprising [E/D/N/Q]-[S/T]-[D/E/Q/N]-[V/L] (SEQ ID NO:5). Optionally,the active peptide comprises an amino acid sequence selected from thegroup consisting of ESDV (SEQ ID NO:6), ESEV (SEQ ID NO:7), ETDV (SEQ IDNO: 8), ETEV (SEQ ID NO:9), DTDV (SEQ ID NO:10), DTEV (SEQ ID NO:11).Optionally, the active peptide has an amino acid sequence comprisingKLSSIESDV (SEQ ID NO:12). Optionally, the active peptide has an aminoacid sequence comprising KLSSIETDV (SEQ ID NO:13). Optionally, thechimeric peptide has an amino acid sequence comprisingFGRKKRRQRRRKLSSIESDV (SEQ ID NO:19) or FGRKKRRQRRRKLSSIETDV (SEQ IDNO:16). Optionally, the chimeric peptide has an amino acid sequenceconsisting of FGRKKRRQRRRKLSSIESDV (SEQ ID NO:19) orFGRKKRRQRRRKLSSIETDV (SEQ ID NO:16). Optionally, the effective dosage isa single dose of 0.05 to 500 mg, optionally 0.1 to 100 mg, 0.5 to 50 mg,or 1-20 mg of the peptide or peptidomimetic. Optionally, the patient hasischemic stroke. Optionally, the patient has hemorrhagic stroke.

Optionally, the patient has above normal susceptibility to side effectsmediated by N-type calcium channels. Optionally, the patient has normalor below normal blood pressure. The invention further provides a methodof assessing potential side effects of an internalization peptide. Themethod involves providing an internalization peptide that promotesuptake of an active peptide that inhibits binding of PSD-95 to an NMDAreceptor into a cell; and determining binding of the internalizationpeptide to an N-type calcium channel. The extent of binding being anindicator of potential side effects in clinical use of theinternalization peptide. Optionally, the internalization peptide isprovided by screening a test peptide to determine whether the testpeptide promotes uptake of the active peptide.

The invention further provides an isolated chimeric agent comprising anactive agent and an internalization peptide that promotes uptake of thechimeric agent into cells. The internalization peptide is a variant ofthe tat peptide YGRKKRRQRRR (SEQ ID NO:1) that has reduced capacity tobind to an N-type calcium channel relative to the tat peptide.Optionally, the active agent is an active agent shown in Table 5.Optionally, the internalization peptide has an amino acid sequencecomprising XGRKKRRQRRR (SEQ ID NO:2), wherein X is an amino acid otherthan Y, or nothing. Optionally, X is F (SEQ ID NO:135). Optionally, X isnothing (SEQ ID NO:136). Optionally, the internalization peptideconsists of GRKKRRQRRRP (SEQ ID NO:3). Optionally, the chimeric agenthas a kD greater than 10 nM for an N-type calcium channel.

The invention further provides a pharmaceutical composition comprisingan isolated chimeric agent as described above and a pharmaceuticallyacceptable carrier.

The invention further provides an internalization peptide having anamino acid sequence comprising XGRKKRRQRRR (SEQ ID NO:2), wherein X isan amino acid other than Y or nothing. Optionally, X is F (SEQ IDNO:135). Optionally, X is nothing (SEQ ID NO:136). Optionally, theinternalization peptide consists of GRKKRRQRRRP (SEQ ID NO:3).Optionally, the internalization peptide has a Kd greater than 10 nM foran N-type calcium channel blocking agent.

The invention further provides in a method of facilitating uptake of anactive agent into a cell comprising contacting the cell with the activeagent linked to an internalization peptide, the improvement wherein theinternalization peptide is screened to determine its capacity to bind toan N-type calcium channel.

The invention further provides in a chimeric agent comprising aninternalization peptide linked to an active agent, the improvementwherein the internalization peptide has an amino acid sequencecomprising XGRKKRRQRRR (SEQ ID NO:2), wherein X is an amino acid otherthan Y, or nothing.

The invention further provides in a method of treating a neurologicaldisease, comprising administering an active agent to a patient having orsusceptible to the disease an effective amount of an active agent havingpharmaceutical activity against the disease, the improvement wherein theactive agent is linked to a tat variant peptide having an amino acidsequence comprising XGRKKRRQRRR (SEQ ID NO:2), wherein X is an aminoacid other than Y, or nothing.

The invention further provides in a method of treating a disease with anactive agent having an intracellular activity effective to treat thedisease, comprising administering an effective amount of the activeagent to a patient having or susceptible to the disease, the improvementwherein the active agent is linked to a tat variant peptide having anamino acid sequence comprising XGRKKRRQRRR (SEQ ID NO:2), wherein X isan amino acid other than Y, or nothing.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, B, C: Results of a receptor binding/inhibition study assessingthe ability of the peptide YGRKKRRQRRRKLSSIESDV (SEQ ID NO:17) toinhibit binding of various radiolabeled ligands to cellular receptors.

FIG. 2: Effect of applying the various peptides on the amplitude ofN-type calcium currents (upper) or whole cell currents (lower) in DRGneurons.

FIGS. 3A and 3B show (A) Effect of Tat-NR2B9c and F-Tat-NR2B9c oncerebral infarction volume in rats treated 1 h after onset of permanentischemia using the pial vessel occlusion model (10 rats/group); and (B)Serial brain sections of a representative rat from each group stainedwith triphenyl-tetrazolium chloride (TTC).

FIG. 4: IC₅₀ determination for certain of the peptides for N-typecalcium currents in DRG neurons.

FIGS. 5A and 5B: Selectivity of Tat-NR2B9c for N-type calcium currentsover L-type currents in DRG neurons. FIG. 5A shows the effect ofTat-NR2B9c (100 μM) and ω-conotoxin (1 μM) on calcium current incultured DGR neurons. FIG. 5B shows the nifedipine inhibition of DRGcalcium current in the presence of Tat-NR2B9c (100 μM intracellular).

FIG. 6: Lack of use-dependence on N-type calcium current inhibition byTat-NR2B9c. Currents were recorded in one representative DRG neuron bydifferent frequency (0.07, 10, 20, 50 Hz). Tat-NR2B9c (100 μM) wasapplied as indicated. The currents shown strong frequency-dependentrundown, and the increase of frequency did not increase Tat-NR2B9c'sinhibition effect.

FIG. 7: Lack of voltage-dependent inhibition by Tat-NR2B9c of N-typecalcium currents. The I-V relationships of Ca²⁺ current in cultured DRGneurons. Tat-NR2B9c (10, 100 μM) was applied in the presence or absenceof 10 μM nifedipine. The currents were elicited using 50 msvoltage-clamp steps from −40 to +50 mV from the holding potential of −60mV.

FIG. 8: Assessment of neuroprotection observed using alternativeC-terminal sequences in the pial occlusion model of permanent ischemiain rats.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

A “chimeric peptide” means a peptide having two component peptides notnaturally associated with one another joined to one another as a fusionprotein or by chemical linkage.

A “fusion polypeptide” refers to a composite polypeptide, i.e., a singlecontiguous amino acid sequence, made up of two (or more) distinct,heterologous polypeptides which are not normally fused together in asingle amino acid sequence.

The term “PDZ domain” refers to a modular protein domain of about 90amino acids, characterized by significant sequence identity (e.g., atleast 60%) to the brain synaptic protein PSD-95, the Drosophila septatejunction protein Discs-Large (DLG), and the epithelial tight junctionprotein ZO1 (ZO1). PDZ domains are also known as Discs-Large homologyrepeats (“DHRs”) and GLGF repeats. PDZ domains generally appear tomaintain a core consensus sequence (Doyle, D. A., 1996, Cell 85:1067-76). Exemplary PDZ domain-containing proteins and PDZ domainsequences disclosed in U.S. application Ser. No. 10/714,537, which isherein incorporated by reference in its entirety.

The term “PL protein” or “PDZ Ligand protein” refers to a naturallyoccurring protein that forms a molecular complex with a PDZ-domain, orto a protein whose carboxy-terminus, when expressed separately from thefull length protein (e.g., as a peptide fragment of 3-25 residues, e.g.3, 4, 5, 8, 9, 10, 12, 14 or 16 residues), forms such a molecularcomplex. The molecular complex can be observed in vitro using the “Aassay” or “G assay” described, e.g., in U.S. application Ser. No.10/714,537, or in vivo.

The term “NMDA receptor,” or “NMDAR,” refers to a membrane associatedprotein that is known to interact with NMDA. The term thus includes thevarious subunit forms described in the Background Section. Suchreceptors can be human or non-human (e.g., mouse, rat, rabbit, monkey).

A “PL motif” refers to the amino acid sequence of the C-terminus of a PLprotein (e.g., the C-terminal 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 20 or25 contiguous residues) (“C-terminal PL sequence”) or to an internalsequence known to bind a PDZ domain (“internal PL sequence”).

A “PL peptide” is a peptide of comprising or consisting of, or otherwisebased on, a PL motif that specifically binds to a PDZ domain.

The terms “isolated” or “purified” means that the object species (e.g.,a peptide) has been purified from contaminants that are present in asample, such as a sample obtained from natural sources that contain theobject species. If an object species is isolated or purified it is thepredominant macromolecular (e.g., polypeptide) species present in asample (i.e., on a molar basis it is more abundant than any otherindividual species in the composition), and preferably the objectspecies comprises at least about 50 percent (on a molar basis) of allmacromolecular species present. Generally, an isolated, purified orsubstantially pure composition comprises more than 80 to 90 percent ofall macromolecular species present in a composition. Most preferably,the object species is purified to essential homogeneity (i.e.,contaminant species cannot be detected in the composition byconventional detection methods), wherein the composition consistsessentially of a single macromolecular species. The term isolated orpurified does not necessarily exclude the presence of other componentsintended to act in combination with an isolated species. For example, aninternalization peptide can be described as isolated notwithstandingthat it is linked to an active peptide.

A “peptidomimetic” refers to a synthetic chemical compound which hassubstantially the same structural and/or functional characteristics of apeptide consisting of natural amino acids. The peptidomimetic cancontain entirely synthetic, non-natural analogues of amino acids, or canbe a chimeric molecule of partly natural peptide amino acids and partlynon-natural analogs of amino acids. The peptidomimetic can alsoincorporate any amount of natural amino acid conservative substitutionsas long as such substitutions also do not substantially alter themimetic's structure and/or inhibitory or binding activity. Polypeptidemimetic compositions can contain any combination of nonnaturalstructural components, which are typically from three structural groups:a) residue linkage groups other than the natural amide bond (“peptidebond”) linkages; b) non-natural residues in place of naturally occurringamino acid residues; or c) residues which induce secondary structuralmimicry, i.e., to induce or stabilize a secondary structure, e.g., abeta turn, gamma turn, beta sheet, alpha helix conformation, and thelike. In a peptidomimetic of a chimeric peptide comprising an activepeptide and an internalization peptide, either the active moiety or theinternalization moiety or both can be a peptidomimetic.

The term “specific binding” refers to binding between two molecules, forexample, a ligand and a receptor, characterized by the ability of amolecule (ligand) to associate with another specific molecule (receptor)even in the presence of many other diverse molecules, i.e., to showpreferential binding of one molecule for another in a heterogeneousmixture of molecules. Specific binding of a ligand to a receptor is alsoevidenced by reduced binding of a detectably labeled ligand to thereceptor in the presence of excess unlabeled ligand (i.e., a bindingcompetition assay).

Excitotoxicity is the pathological process by which neurons are damagedand killed by the overactivation of receptors for the excitatoryneurotransmitter glutamate, such as the NMDA receptors such as NMDAR 2B.

A standard tat internalization peptide comprises the amino acid sequenceYGRKKRRQRRR (SEQ ID NO:1).

A variant tat internalization peptide has at least one amino aciddeleted substituted, or internally inserted relative to a standard tatpeptide.

An active agent is used to describe a compound that has or may have apharmacological activity. Agents include compounds that are known drugs,compounds for which pharmacological activity has been identified butwhich are undergoing further therapeutic evaluation, and compounds thatare members of collections and libraries that are to be screened for apharmacological activity. An active peptide is an active agent that is apeptide. An active chimeric agent comprises an active agent linked to aninternalization peptide.

A “pharmacological” activity means that an active agent exhibits anactivity in a screening system that indicates that the active agent isor may be useful in the prophylaxis or treatment of a disease. Thescreening system can be in vitro, cellular, animal or human. Agents canbe described as having pharmacological activity notwithstanding thatfurther testing may be required to establish actual prophylactic ortherapeutic utility in treatment of a disease.

Statistically significant refers to a p-value that is <0.05, preferably<0.01 and most preferably <0.001.

II. General

The invention provides chimeric peptides and peptidomimetics thereofuseful for reducing damaging effects of stroke and other neurologicalconditions mediated at least in part by NMDAR excitotoxicity. Thechimeric peptides have at least two components. The first component isan active peptide having an amino acid sequence including or based onthe PL motif of a NMDA Receptor 2 subunit (e.g., GenBank accessionnumber 4099612 for NMDA NR2B) (i.e., a PL peptide). Although anunderstanding of mechanism is not required for practice of theinvention, it is believed that such peptides act at least in part byinhibiting interaction between NMDARs with postsynaptic density 95protein (i.e., PSD-95 inhibitors).

The active peptides may also inhibit interactions between PSD-95 andnNOS and other glutamate receptors (e.g., kainite receptors or AMPAreceptors). Unlike glutamate antagonists that have previously failedclinical trials, such peptides can disrupt neurotoxic signaling duringischemia without incurring the negative consequences of loss of otherfunctions of NMDARs. The second component of the chimeric peptide is aninternalization peptide that represents a variant of the standard tatpeptide fused to NMDAR 2B peptides in previous work.

The use of a variant tat peptide is premised in part on the resultsdescribed in the present application that a standard tat peptide,particularly when joined to an NMDAR peptide KLSSIESDV (SEQ ID NO:12),binds to N-type calcium channels and inhibits their activity. N-typecalcium channels located on presynaptic nerve terminals regulateneurotransmitter release, including that from the spinal terminations ofprimary afferent nocioceptors. The pharmacological effects of binding toN-type channels have been well characterized in connection with the drugZiconotide (or Prialt, a synthetic form of the cone snail peptideomega-conotoxin M-VII-A precursor). Binding to N-type calcium channelshas been associated with numerous activities, some or all of which maybe undesirable in stroke patients. These activities include analgesiamuch stronger than that induced by morphine, hypotension, decreasedlevels of consciousness, depression, cognitive impairment,hallucination, elevation of creatine kinase levels, and urinaryretention (see, e.g., Brose et al., Clin J Pain 13: 256-259, (1997);Mathur et al., Semin Anesthesia Perioperative Med Pain 19: 67-75, 2000,Staats et al., JAMA 291: 63-70, 2004, McGuire et al., J CardiovascPharmacol 30: 400-403, 1997. The Mayo clinic lists the following sideeffects observed after treatment with Prialt, which is highly selectivefor N-type calcium channels.

TABLE 1 Severity Incidence Phenotypes Serious Common Seeing, hearing, orfeeling things that are not there; thoughts of killing oneself. LessChest pain; chills; confusion; convulsions; cough; dark-colored Commonurine; dizziness; drowsiness; fainting; fast heartbeat; fever; generalfeeling of illness; lightheadedness; muscle spasm or jerking of allextremities; muscle stiffness; rapid, shallow breathing; shortness ofbreath; sneezing; sore throat; stiff neck or back; tightness in chest;troubled breathing; trouble concentration; trouble sleeping; unusualtiredness or weakness; wheezing. Overdose Decreased awareness orresponsiveness; severe sleepiness; shakiness and unsteady walk;trembling or other problems with muscle control or coordination;uncontrolled eye movements; unsteadiness Moderate Common Burning; changein walking and balance; clumsiness or unsteadiness; confusion; crawlingfeelings; diarrhea; dizziness; excessive muscle tone, muscle tension ortightness; fear; feeling of constant movement of self or surroundings;fever; headache; itching; lack or loss of strength; lightheadedness;loss of appetite; nausea; nervousness; numbness; problems with speech orspeaking; sensation of spinning; trembling, or other problems withmuscle control or coordination; uncontrolled eye movements; urinaryretention; vomiting; weight loss. Less Acid or sour stomach; back pain;bad, unusual or unpleasant Common (after)taste; belching; bladder pain;bloody or cloudy urine; bruising; cerebrospinal fluid abnormal; changein taste; congestion; constipation; continuing ringing or buzzing orother unexplained noise in ears; crying; decreased awareness orresponsiveness; dehydration; depersonalization; depression; difficult,burning or painful urination; difficulty in moving; difficulty seeing atnight; double vision; dry mouth; dry skin; dryness or soreness ofthroat; dysphoria; euphoria; fainting; fast or irregular heartbeat;feeling that others can hear your thoughts, are watching you, or cancontrol your behavior; frequent urge to urinate; hearing loss;heartburn; hoarseness; hostility; increased sensitivity of eyes tosunlight; increased sensitivity to pain or touch; indigestion; loss ofbladder control; loss of memory or problems with memory; lung disorder;neck pain; nerve pain; pain in joints; pale skin; pounding in ears;quick to react or overreact emotionally; rapidly changing moods; red,scaly, swollen or peeling areas of skin; redness or pain at cathetersite; runny nose; severe muscle stiffness; sleeplessness; slow or fastheartbeat; stomach discomfort, upset or pain; sweating; swelling orredness in joints; tender, swollen glands in neck; trouble inswallowing; unusual bleeding or bruising; unusual tiredness or weakness;voice changes; warmth on skin; weakness or heaviness in legs.

The present chimeric peptides and peptidomimetics have reduced oreliminated binding to and inhibiting of N-type calcium channels comparedwith Tat-NR2B9c and thus avoid the large number of side effects observedwith highly specific inhibitors of the N-type calcium channel, includingsevere psychiatric side effects. The reduction in side effects resultsin an increase in the therapeutic index for treatment of humans of thepresent chimeric peptide and peptidomimetics relative to Tat-NR2B9c.

The present inventors have further found that binding to N-type calciumchannels can be avoided by uses of variants of the standard tatsequence. The combination of a tat variant and an active peptide basedon or including the C-terminus of NMDAR 2B or other subtype allowstreatment of stroke with reduced side effects due to inhibition ofN-type calcium channels.

III. Active Peptides

Active peptides useful in the invention inhibit interaction between PDZdomains 1 and 2 of postsynaptic density-95 protein (PSD-95) (human aminoacid sequence provided by Stathakism, Genomics 44(1):71-82 (1997)) andthe C-terminal PL sequence of one or more NMDA Receptor 2 subunitsincluding the NR2B subunit of the neuronal N-methyl-D-aspartate receptor(Mandich et al., Genomics 22, 216-8 (1994)). NMDAR2B has GenBank ID4099612, a C-terminal 20 amino acids FNGSSNGHVYEKLSSIESDV (SEQ ID NO:24)and a PL motif ESDV (SEQ ID NO:6). Active peptides preferably inhibitthe human forms of PSD-95 and human NMDAR receptors. However, inhibitioncan also be shown from species variants of the proteins. A list of NMDAand glutamate receptors that can be used appears below:

TABLE 2 NMDA RECEPTORS WITH PL SEQUENCES C-terminal C-terminal internalName GI or Acc# 20 mer sequence 4 mer sequence PL? PL ID NMDAR1 307302HPTDITGPLNLSDPSVSTVV STVV X AA216 (SEQ ID NO:25) (SEQ ID NO:39) NMDAR1-1292282 HPTDITGPLNLSDPSVSTVV STVV X AA216 (SEQ ID NO:25) (SEQ ID NO:39)NMDAR1-4 472845 HPTDITGPLNLSDPSVSTVV STVV X AA216 (SEQ ID NO:25) (SEQ IDNO:39) NMDAR1-3b 2343286 HPTDITGPLNLSDPSVSTVV STVV X AA216 (SEQ IDNO:25) (SEQ ID NO:39) NMDAR1-4b 2343288 HPTDITGPLNLSDPSVSTVV STVV XAA216 (SEQ ID NO:25) (SEQ ID NO:39) NMDAR1-2 11038634RRAIEREEGQLQLCSRHRES HRES (SEQ ID NO:26) (SEQ ID NO:40) NMDAR1-311038636 RRAIEREEGQLQLCSRHRES HRES (SEQ ID NO:26) (SEQ ID NO:40) NMDAR2C6006004 TQGFPGPCTWRRISSLESEV ESEV X AA180 (SEQ ID NO:27) (SEQ ID NO:7)NMDAR3 560546 FNGSSNGHVYEKLSSIESDV ESDV X AA34.1 (SEQ ID NO:24) (SEQ IDNO:6) NMDAR3A 17530176 AVSRKTELEEYQRTSRTCES TCES (SEQ ID NO:28) (SEQ IDNO:41) NMDAR2B 4099612 FNGSSNGHVYEKLSSIESDV ESDV X (SEQ ID NO:24) (SEQID NO:6) NMDAR2A 558748 LNSCSNRRVYKKMPSIESDV ESDV X AA34.2 (SEQ IDNO:29) (SEQ ID NO:6) NMDAR2D 4504130 GGDLGTRRGSAHFSSLESEV ESEV X (SEQ IDNO:30) (SEQ ID NO:7) Glutamate AF009014 QPTPTLGLNLGNDPDRGTSI GTSI Xreceptor (SEQ ID NO:31) (SEQ ID NO:42) delta 2 Glutamate I28953MQSIPCMSHSSGMPLGATGL ATGL X receptor 1 (SEQ ID NO:32) (SEQ ID NO:43)Glutamate L20814 QNFATYKEGYNVYGIESVKI SVKI X receptor 2 (SEQ ID NO:33)(SEQ ID NO:44) Glutamate AF167332 QNYATYREGYNVYGTESVKI SVKI X receptor 3(SEQ ID NO:34) (SEQ ID NO:44) Glutamate U16129 HTGTAIRQSSGLAVIASDLP SDLPreceptor 4 (SEQ ID NO:35) (SEQ ID NO:45) Glutamate U16125SFTSILTCHQRRTQRKETVA ETVA X receptor 5 (SEQ ID NO:36) (SEQ ID NO:46)Glutamate U16126 EVINMHTFNDRRLPGKETMA ETMA X receptor 6 (SEQ ID NO:37)(SEQ ID NO:47) Glutamate U16127 RRLPGKDSMACSTSLAPVFP PVFP receptor 7(SEQ ID NO:38) (SEQ ID NO:48)

Evidence for a role of different NMDAR subtypes in excitotoxicity isprovided by e.g., Lynch, J. Pharm. Exp. Therapeutics 300, 717-723(2002); Kemp, Nature Neurosci. supplement, vol 5 (2002). Some activepeptides inhibit interactions between PSD-95 and multiple NMDARsubunits. In such instances, use of the peptide does not necessarilyrequire an understanding of the respective contributions of thedifferent NMDARs to excitotoxicity. Other active peptides are specificfor a single NMDAR.

Active peptides include or are based on a PL motif from the C-terminusof any of the above subunits and have an amino acid sequence comprising[S/T]-X-[V/L] (SEQ ID NO:14). This sequence preferably occurs at theC-terminus of the peptides of the invention. Preferred peptides have anamino acid sequence comprising [E/D/N/Q]-[S/T]-[D/E/QIN]-[V/L] (SEQ IDNO:5) at their C-terminus. Exemplary peptides comprise: ESDV (SEQ IDNO:6), ESEV (SEQ ID NO.:7), ETDV (SEQ ID NO:8), ETEV (SEQ ID NO:9), DTDV(SEQ ID NO:10), and DTEV (SEQ ID NO:11) as the C-terminal amino acids.Two particularly preferred peptides are KLSSIESDV (SEQ ID NO:12), andKLSSIETDV (SEQ ID NO:13). Peptides of the invention without aninternalization peptide usually have 3-25 amino acids, peptide lengths(also without an internalization peptide) of 5-10 amino acids, andparticularly 9 amino acids are preferred. In some such active peptides,all amino acids are from the C-terminus of an NMDA receptor.

Other active peptides include PDZ domain 1 and/or 2 of PSD-95 or asubfragment of any of these that inhibits interactions between PSD-95and an NMDA receptor, such as NMDA 2B. Such active peptides comprise atleast 50, 60, 70, 80 or 90 amino acids from PDZ domain 1 and/or PDZdomain 2 of PSD-95, which occur within approximately amino acids 65-248of PSD-95 provided by Stathakism, Genomics 44(1):71-82 (1997) (humansequence) or NP_(—)031890.1, GI:6681195 (mouse sequence) orcorresponding regions of other species variants.

III. Internalization Peptides

Any of the active peptides of the invention can be linked, preferably atits N-terminus, to an internalization peptide that facilitatestranslocation through the plasma membrane of a cell. Internalizationpeptides comprise a variant of a standard tat sequence YGRKKRRQRRR (SEQID NO:1). Although practice of the invention is not dependent on anunderstanding of mechanism, it is believed that both capacity to crossmembranes and binding to N-type calcium channels are conferred by theunusually high occurrence of positively charged residues Y, R and K inthe peptide. Variant peptides for use in the invention should retainability to facilitate uptake into cells but have reduced capacity tobind N-type calcium channels. Some suitable internalization peptidescomprise or consist of an amino acid sequence XGRKKRRQRRR (SEQ ID NO:2),in which X is an amino acid other than Y (e.g., any of the other 19natural amino acids) or nothing (in which case G is a free N-terminalresidue). A preferred tat variant has the N-terminal Y residuesubstituted with F. Thus, a tat variant comprising or consisting ofFGRKKRRQRRR (SEQ ID NO:49) is preferred. Another preferred variant tatinternalization peptide consists of GRKKRRQRRR (SEQ ID NO:50). Ifadditional residues flanking XGRKKRRQRRR (SEQ ID NO:2) are present(beside the active peptide) the residues can be for example, naturalamino acids flanking this segment from a tat protein, spacer or linkeramino acids of a kind typically used to join two peptide domains, e.g.,Gly (Ser)₄ (SEQ ID NO:134), T G E K P (SEQ ID NO:51), GGRRGGGS (SEQ IDNO:52), or LRQRDGERP (SEQ ID NO:53) (see, e.g., Tang et al. (1996), J.Biol. Chem. 271, 15682-15686; Hennecke et al. (1998), Protein Eng. 11,405-410)), or can be any other amino acids that do not detectably reducecapacity to confer uptake of the variant without the flanking residuesand do not significantly increase inhibition of N-type calcium channelsrelative to the variant without the flanking residues. Preferably, thenumber of flanking amino acids other than an active peptide does notexceed ten on either side of XGRKKRRQRRR (SEQ ID NO:2). Preferably, noflanking amino acids are present, and the internalization peptide islinked at its C-terminus directly to an active peptide.

Other internalization peptides of the invention that can be used toallow uptake of any of the active peptides of the invention forinhibition of PSD-95 interactions without inhibiting N-type calciumchannels include those presented in Table 3 below. It is recommendedthat these internalization peptides be screened to confirm desireduptake and lack of inhibition of N-type calcium channels, as describedin the Examples.

The data presented in the examples demonstrate that mutation of theN-terminal tyrosine residue (Y) of Tat-NR2B9c to phenylalanine (F) issufficient to abrogate inhibition of the N-type calcium channel withoutreducing the ability of the remainder of the peptide to localize to thesite of action for this drug in the brain and reduce the damagefollowing induced stroke in animals models of permanent ischemia.Further, the experiments demonstrate that Tat alone (YGRKKRRQRRR (SEQ IDNO:1)) is sufficient to induce the observed inhibition of the N-typecalcium channel, and that different peptides added at the C-terminushave only a mild effect on the inhibition when attached to Tat. Thus,change or removal of the tyrosine at the N-terminus of the Tat sequenceis likely to be important to reduction of binding. Mutation of basicamino acid residues near this tyrosine can also result in a reduction ofbinding to and inhibition of N-type calcium channels. The exemplarysequences in the table below are predicted herein to maintain transportcapability without inhibiting N-type calcium channels and thus allow agreater therapeutic index for the treatment of stroke or neurotrauma.

TABLE 3 X-FGRKKRRQRRRKLSSIESDV SEQ ID NOS:19, 77, 78, 79 (F-TatNR2B9c)X-GKKKKKQKKKKLSSIESDV SEQ ID NO:54, 80, 81, 82 X-RKKRRQRRRKLSSIESDV SEQID NO:55, 83, 84, 85 X-GAKKRRQRRRKLSSIESDV SEQ ID NO:56, 86, 87, 88X-AKKRRQRRRKLSSIESDV SEQ ID NO:57, 89, 90, 91 X-GRKARRQRRRKLSSIESDV SEQID NO:58, 92, 93, 94 X-RKARRQRRRKLSSIESDV SEQ ID NO:59, 95, 96, 97X-GRKKARQRRRKLSSIESDV SEQ ID NO:60, 98, 99, 100 X-RKKARQRRRKLSSIESDV SEQID NO:61, 101, 102, 103 X-GRKKRRQARRKLSSIESDV SEQ ID NO:62, 104, 105,106 X-RKKRRQARRKLSSIESDV SEQ ID NO:63, 107, 108, 109X-GRKKRRQRARKLSSIESDV SEQ ID NO:64, 110, 111, 112 X-RKKRRQRARKLSSIESDVSEQ ID NO:65, 113, 114, 115 X-RRPRRPRRPRRKLSSIESDV SEQ ID NO:66, 116,117, 118 X-RRARRARRARRKLSSIESDV SEQ ID NO:67, 119, 120, 121X-RRRARRRARRKLSSIESDV SEQ ID NO:68, 122, 123, 124 X-RRRPRRRPRRKLSSIESDVSEQ ID NO:69, 125, 126, 127 X-RRPRRPRRKLSSIESDV SEQ ID NO:70, 128, 129,130 X-RRARRARRKLSSIESDV SEQ ID NO:71, 131, 132, 133

X can represent a free amino terminus, a biotin molecule or othercapping moiety including, but not limited to, H, acetyl, benzoyl, alkylgroup (aliphatic), pyroglutamate, alkyl group with cycloalkyl group atthe end, biotin with alkyl spacer, (5,6)-FAM. Chemical coupling of thecapping group to the N-terminal peptide can be through an amidechemistry, sulphamide chemistry, sulphone chemistry, alkylationchemistry. In addition, X can also be an amino acid other that tyrosine.

Internalization peptides are usually linked to active peptides as fusionpeptides, but can also be joined by chemical linkage. Coupling of thetwo constituents can be accomplished via a coupling or conjugatingagent. Numerous such agents are commercially available and are reviewedby S. S. Wong, Chemistry of Protein Conjugation and Cross-Linking, CRCPress (1991). Some examples of cross-linking reagents includeJ-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) orN,N′-(1,3-phenylene) bismaleimide; N,N′-ethylene-bis-(iodoacetamide) orother such reagent having 6 to 11 carbon methylene bridges (whichrelatively specific for sulfhydryl groups); and1,5-difluoro-2,4-dinitrobenzene (which forms irreversible linkages withamino and tyrosine groups). Other cross-linking reagents includep,p′-difluoro-m,m′-dinitrodiphenylsulfone (which forms irreversiblecross-linkages with amino and phenolic groups); dimethyl adipimidate(which is specific for amino groups); phenol-1,4-disulfonylchloride(which reacts principally with amino groups); hexamethylenediisocyanateor diisothiocyanate, or azophenyl-p-diisocyanate (which reactsprincipally with amino groups); glutaraldehyde (which reacts withseveral different side chains) and disdiazobenzidine (which reactsprimarily with tyrosine and histidine).

Peptides such as those just described can optionally be derivatized(e.g., acetylated, phosphorylated and/or glycoslylated) to improve thebinding affinity of the inhibitor, to improve the ability of theinhibitor to be transported across a cell membrane or to improvestability. As a specific example, for inhibitors in which the thirdresidue from the C-terminus is S or T, this residue can bephosphorylated before use of the peptide.

Peptides of the invention, optionally fused to internalization domains,can be synthesized by solid phase synthesis or recombinant methods.Peptidomimetics can be synthesized using a variety of procedures andmethodologies described in the scientific and patent literature, e.g.,Organic Syntheses Collective Volumes, Gilman et al. (Eds) John Wiley &Sons, Inc., NY, al-Obeidi (1998) Mol. Biotechnol. 9:205-223; Hraby(1997) Curr. Opin. Chem. Biol. 1:114-119; Ostergaard (1997) Mol. Divers.3:17-27; Ostresh (1996) Methods Enzymol. 267:220-234.

V. N-Type Calcium Channels

N-type calcium channels are hetero-oligomeric complexes consisting ofα_(1B)-(Cav2.2), β-, and α₂δ-subunits and sometimes y subunits. Theα_(1B)-subunit forms the main channel and is encoded by a single gene.There are four α₂δ-subunit genes (α₂δ-1-α₂δ-4)(Snutch et al., Molecularproperties of voltage-gated calcium channels. In: Voltage-gated calcium(Zamponi G, ed), pp 61-94. New York: Landes Bioscience, 2005. Catterall,Biochemical studies of Ca²⁺ channels. In: Voltage-Gated Calcium (ZamponiG, ed), pp 48-60. New York: Landes Bioscience, 2005). There is closeconservation of N-type calcium channels across species. Thus tatvariants can be screened for lack of binding using N-type calciumchannels from humans or other species, such as rats.

The α_(1B)-subunit N-type calcium channel described by Williams et al.,1992 (Science 257 (5068), 389-395 (1992), Genebank Acc No. Q00975,Species: Homo Sapiens) and Coppola et al., 1994 (FEBS Lett. 338 (1), 1-5(1994), Genebank Acc No 055017, Species: Mus Musculus) and Dubel et al.,1992 (Proc. Natl. Acad. Sci. U.S.A. 89 (11), 5058-5062 (1992) GenebankAcc No Q02294, Species: Rattus norvegicus) including splice variants andfragments thereof having similar calcium channel activity to the intactprotein is preferred for screening. Allelic variants and speciesvariants having at least 90% sequence identity with any of the abovesequences can also be used. Optionally, the α_(1B) subunit can be usedin combination with an alpha2(a-e)/delta, beta1-4, and/or gamma subunit.

VI. Screening Methods

1. Measuring Binding to N-Type Calcium Channels

Internalization peptides can be screened for binding to N-type calciumchannels by a competitive binding assay using a labeled peptide known tobind such channels (e.g., Ziconotide). The N-type calcium channel can beprovided in purified form or naturally or recombinantly expressed fromcells. If provided in purified form, the N-type calcium channel can beimmobilized to beads or to a microtiter well. The amount of label boundto the calcium channel after incubation with the labeled peptide andinternalization peptide under test is inversely related to the capacityof the internalization peptide under test to bind to the calciumchannel. The assay can be performed on a high throughput basis in thewells of a microtiter plate. Negative and positive controls can also beincluded. A negative control can be vehicle. A positive control can beunlabelled form of the peptide known to bind N-type calcium channels.Preferably an internalization peptide has a Kd greater than 10 nM,preferably greater than 100 nM for an N-type calcium channel.

2. Measuring Inhibition of N-type Calcium Channels

Internalization peptides and chimeric agents comprising aninternalization peptide linked to an active agent, such as an activepeptide can be screened for their capacity to inhibit ionic currentsmediated by N-type calcium channels. Inhibition means a statisticallysignificant reduction in the measured ionic current carried by N-typecalcium channels. Such a reduction should be greater than a 20%reduction in measured current, preferably greater than 30% reduction,and more preferably greater than 40% reduction. Inhibition can bedetermined by performing whole-cell patch clamp recordings in dorsalroot ganglion neurons, in which N-type calcium currents are expressed.Cultures of dorsal root ganglions (DRGs) were prepared from Swiss miceat 13-14 days of gestation. In brief, DRG's are dissected and subjectedto trypsin digestion for 20 min at 37° C., mechanically dissociated andplated on cover slips coated with poly-D-lysine. They are grown in serumfree MEM (Neurobasal MEM, B-27—Gibco Invitrogen Corporation, Carlsbad,Calif.). After 3-5 days, 10 μM FUDR solution is added to inhibit glialproliferation. The cultures are maintained at 37° C. in a humidified 5%CO₂ atmosphere and fed twice a week. Whole-cell recording is carried outat room temperature 10-14 days after plating. Electrophysiologyrecordings: Whole-cell recordings are performed with an Axopatch-1Bamplifier (Axon Instruments, Foster City, Calif.) in the voltage-clampmode. Recording electrodes, with resistances of 3-5 MΩ, are constructedfrom thin-walled borosilicate glass (1.5 mm diameter; World PrecisionInstruments, Sarasota, Fla.) using a two-stage puller (PP83; Narishige,Tokyo, Japan). Data are digitized, filtered (2 kHz), and acquiredon-line using the programs of pClamp 9 (Axon Instruments). The pipettesare filled with a solution containing (mM): CsCl 110, MgCl2 3, EGTA 10,HEPES 10, MgATP 3, GTP 0.6. The pH is adjusted to 7.2 with CsOH. Thebath solution contained (mM): CaCl2 1, BaCl2 10, HEPES 10, TEA-Cl 160,Glucose 10, TTX 0.0002 at pH (NaOH) 7.4. Whole-cell currents areelicited using 40 ms depolarizing pulses to +20 mV from a holdingpotential of −60 mV, applied every 15 s. To test the use-dependentinhibition, currents are elicited using 10 ms depolarizing pulses to +20mV from a holding potential of −60 mV, applied every 0.02 s (50 Hz),0.05 s (20 Hz), 0.1 s (10 Hz) or 15 s (0.07 Hz) respectively.

3. Measuring Internalization Activity

Variants of the tat internalization peptide can be tested for transportactivity in an animal. Internalization peptides can be tested alone orwhen linked to an active agent, such an active peptide, e.g., KLSSIESDV(SEQ ID NO:12). The internalization peptide, optionally linked to anactive agent, such as a peptide, is labeled, preferably with afluorescent label, such as dansyl chloride. The internalization peptideis then injected peripherally into an animal, such as a mouse.Intraperitoneal or intravenous injection is suitable, for example. Aboutan hour after injection, the mice are sacrificed, perfused with fixativesolution (3% paraformaldehyde, 0.25% glutaraldehyde, 10% sucrose, 10U/mL heparin in saline). Brains are then removed, frozen and sections.Sections are analyzed for fluorescence using a confocal microscope.Internalization activity is determined from fluorescence, optionallyrelative to positive and negative controls. A suitable positive controlis the standard tat peptide linked to the same active peptide (ifpresent) as the internalization peptide under test. A suitable negativecontrol is fluorescently labeled active peptide not linked to aninternalization peptide. Unlabelled vehicle can also be used as anegative control.

Similar experiments can be performed in cell culture to test tatvariants (see US20030050243). A variant fluorescently labeled tatpeptide, optionally linked to an active peptide is applied to a corticalneuronal culture. Uptake is determined using fluorescence microscopyover several minutes after application. Increased uptake can bedetermined relative to positive and negative controls as described forthe experiments on uptake in an animal.

4. Measuring Activity in Treating Stroke

The activity of chimeric peptides comprising a internalization peptidelinked to an active peptide (or a peptidomimetic of such a chimericpeptide) can be tested in various animal models of stroke. In one suchmodel, in adult male Sprague-Dawley rats subjected to transient middlecerebral artery occlusion (MCAO) for 90 minutes by the intraluminalsuture method (36,37). Animals are fasted overnight and injected withatropine sulfate (0.5 mg/kg IP). After 10 minutes anesthesia is induced.Rats are orally intubated, mechanically ventilated, and paralyzed withpancuronium bromide (0.6 mg/kg IV). Body temperature is maintained at36.5-37.5° C. with a heating lamp. Polyethylene catheters in the femoralartery and vein are used to continuously record blood pressure and tosample blood for gas and pH measurements. Transient MCAO is achieved for90 min by introducing a poly-L-lysine-coated 3-0 monofilament nylonsuture (Harvard Apparatus) into the circle of Willis via the internalcarotid artery, effectively occluding the middle cerebral artery. Thisproduces an extensive infarction encompassing the cerebral cortex andbasal ganglia. Animals are treated with either a chimeric peptide undertest or a negative or positive control. Treatment can be either beforeor up to one hour after inducing ischemia. A negative control can bevehicle. A positive control can be the Tat-NR2B9c peptide,YGRKKRRQRRRKLSSIESDV (SEQ ID NO:17), previously shown to be effective.The chimeric peptide is delivered by a single intravenous bolusinjection 45 min prior to MCAO (3 mmoles/g). After administeringcompounds to the animals, infarction volume and/or disability index aredetermined. Infarction volumes are usually determined 24 hr posttreatment but can be determined at a later time such as 3, 7, 14 or 60days. Disability index can be monitored over time, e.g., at 2 hr posttreatment, 24 hr post treatment, one week and one month post treatment.Chimeric peptides showing a statistically significant reduction ininfarction volume and/or disability index relative to control animalsnot treated with the compounds are identified as having activity usefulfor practicing the methods of the invention.

Similar experiments can be performed in animal subject to permanentischemia. Permanent ischemia of the middle cerebral artery pial vesselcan be carried out as described by Forder et al., Am J Physiol HeartCirc Physiol 288:H1989-H1996 (2005). In brief, the right ECA iscannulated with PE 10 polyethylene tubing. The skull is exposed via amidline incision, and a 6- to 8-mm cranial window is made over the rightsomatosensory cortex (2 mm caudal and 5 mm lateral to bregma). The pialarteries are visualized by injecting small boluses (10-20 μL) of thevital dye patent blue violet (10 mMol/L; Sigma) in normal saline, intothe ECA. The same three pial arteriolar MCA branches are electricallycauterized and dye injections are repeated to ensure the interruption offlow through the cauterized arterioles. The incision is then closed andthe animal returned to its cage and allowed free access to food andwater. This permanent ischemia model produces a highly reproduciblesmall infarction limited to the cortex underlying the coagulatedterminal pial arteries.

The left middle cerebral artery can be occluded by the intraluminalsuture method described by Longa, Stroke 20, 84-91 (1989). In brief, theleft common carotid artery (CCA) is exposed through a midline neckincision and is dissected free from surrounding nerves and fascia, fromits bifurcation to the base of the skull. The occipital artery branchesof the external carotid artery (ECA) are then isolated, and thesebranches dissected and coagulated. The ECA is dissected further distallyand coagulated along with the terminal lingual and maxillary arterybranches, which are then divided. The internal carotid artery (ICA) isisolated and separated from the adjacent vagus nerve, and thepterygopalatine artery is ligated close to its origin. The tip of a 4-cmlength of 3-0 monofilament nylon suture (Harvard Apparatus) is roundedby burning to achieve a tip diameter of 0.33-0.36 mm and tip length of0.5-0.6 mm and coated with poly-L-lysine (Belayev et al., 1996). Thesuture is introduced through the CCA and advanced into the ICA andthence into the circle of Willis (about 18-20 mm from the carotidbifurcation), effectively occluding the middle cerebral artery. The silksuture around the CCA is tightened around the intraluminal nylon sutureto secure it and permanently occlude the middle cerebral artery.

5. Cell-Based Screening of Active Peptides

Optionally, active peptides or peptidomimetics thereof can also bescreened for capacity to inhibit interactions between PSD-95 and NMDAR2B using assays described in e.g., US 20050059597. Useful peptidestypically have IC50 values of less than 50 uM, 25 μM, 10 μM, 0.1 μM or0.01 μM in such an assay. Preferred peptides typically have an IC50value of between 0.001-1 μM, and more preferably 0.05-0.5 or 0.05 to 0.1μM.

VI. Stroke and Related Conditions

A stroke is a condition resulting from impaired blood flow in the CNSregardless of cause. Potential causes include embolism, hemorrhage andthrombosis. Some neuronal cells die immediately as a result of impairedblood flow. These cells release their component molecules includingglutamate, which in turn activates NMDA receptors, which raiseintracellular calcium levels, and intracellular enzyme levels leading tofurther neuronal cell death (the excitotoxicity cascade). The death ofCNS tissue is referred to as infarction. Infarction Volume (i.e., thevolume of dead neuronal cells resulting from stroke in the brain) can beused as an indicator of the extent of pathological damage resulting fromstroke. The symptomatic effect depends both on the volume of aninfarction and where in the brain it is located. Disability index can beused as a measure of symptomatic damage, such as the Rankin StrokeOutcome Scale (Rankin, Scott Med J; 2:200-15 (1957)) and the BarthelIndex. The Rankin Scale is based on assessing directly the globalconditions of a patient as follows.

TABLE 4 0 No symptoms at all 1 No significant disability despitesymptoms; able to carry out all usual duties and activities. 2 Slightdisability; unable to carry out all previous activities but able to lookafter own affairs without assistance. 3 Moderate disability requiringsome help, but able to walk without assistance 4 Moderate to severedisability; unable to walk without assistance and unable to attend toown bodily needs without assistance. 5 Severe disability; bedridden,incontinent, and requiring constant nursing care and attention.

The Barthel Index is based on a series of questions about the patient'sability to carry out 10 basic activities of daily living resulting in ascore between 0 and 100, a lower score indicating more disability(Mahoney et al., Maryland State Medical Journal 14:56-61 (1965)).

Alternatively stroke severity/outcomes can be measured using the NIHstroke scale, available at world wide webninds.nih.gov/doctors/NIH_Stroke_Scale_Booklet.pdf. The scale is basedon the ability of a patient to carry out 11 groups of functions thatinclude assessments of the patient's level of consciousness, motor,sensory and language functions.

An ischemic stroke refers more specifically to a type of stroke thatcaused by blockage of blood flow to the brain. The underlying conditionfor this type of blockage is most commonly the development of fattydeposits lining the vessel walls. This condition is calledatherosclerosis. These fatty deposits can cause two types ofobstruction. Cerebral thrombosis refers to a thrombus (blood clot) thatdevelops at the clogged part of the vessel “Cerebral embolism” refersgenerally to a blood clot that forms at another location in thecirculatory system, usually the heart and large arteries of the upperchest and neck. A portion of the blood clot then breaks loose, entersthe bloodstream and travels through the brain's blood vessels until itreaches vessels too small to let it pass. A second important cause ofembolism is an irregular heartbeat, known as arterial fibrillation. Itcreates conditions in which clots can form in the heart, dislodge andtravel to the brain. Additional potential causes of ischemic stroke arehemorrhage, thrombosis, dissection of an artery or vein, a cardiacarrest, shock of any cause including hemorrhage, and iatrogenic causessuch as direct surgical injury to brain blood vessels or vessels leadingto the brain or cardiac surgery. Ischemic stroke accounts for about 83percent of all cases of stroke.

Transient ischemic attacks (TIAs) are minor or warning strokes. In aTIA, conditions indicative of an ischemic stroke are present and thetypical stroke warning signs develop. However, the obstruction (bloodclot) occurs for a short time and tends to resolve itself through normalmechanisms.

Hemorrhagic stroke accounts for about 17 percent of stroke cases. Itresults from a weakened vessel that ruptures and bleeds into thesurrounding brain. The blood accumulates and compresses the surroundingbrain tissue. The two general types of hemorrhagic strokes areintracerebral hemorrhage and subarachnoid hemorrhage. Hemorrhagic strokeresult from rupture of a weakened blood vessel ruptures. Potentialcauses of rupture from a weakened blood vessel include a hypertensivehemorrhage, in which high blood pressure causes a rupture of a bloodvessel, or another underlying cause of weakened blood vessels such as aruptured brain vascular malformation including a brain aneurysm,arteriovenous malformation (AVM) or cavernous malformation. Hemorrhagicstrokes can also arise from a hemorrhagic transformation of an ischemicstroke which weakens the blood vessels in the infarct, or a hemorrhagefrom primary or metastatic tumors in the CNS which contain abnormallyweak blood vessels. Hemorrhagic stroke can also arise from iatrogeniccauses such as direct surgical injury to a brain blood vessel. Ananeurysm is a ballooning of a weakened region of a blood vessel. If leftuntreated, the aneurysm continues to weaken until it ruptures and bleedsinto the brain. An arteriovenous malformation (AVM) is a cluster ofabnormally formed blood vessels. A cavernous malformation is a venousabnormality that can cause a hemorrhage from weakened venous structures.Any one of these vessels can rupture, also causing bleeding into thebrain. Hemorrhagic stroke can also result from physical trauma.Hemorrhagic stroke in one part of the brain can lead to ischemic strokein another through shortage of blood lost in the hemorrhagic stroke.

Several other neurological conditions can also result in neurologicaldeath through NDMAR-mediated excitotoxicity. These conditions includeepilepsy, hypoxia, traumatic injury to the CNS not associated withstroke such as traumatic brain injury and spinal cord injury,Alzheimer's disease and Parkinson's disease.

VII. Methods of Treatment

The chimeric peptides described above or peptidomimetics thereof areused to treat patients with stroke. Treatment is usually initiated assoon as possible after initiation of the stroke. Occasionally, treatmentcan be initiated at or before onset of stroke in patients known to be athigh risk. Risk factors include hypertension, diabetes, family history,smoking, previous stroke, and undergoing surgery. Usually, treatment isfirst administered within one to 24 hours after initiation of stroke.Often a single dose of chimeric peptide of the invention is sufficient.However, multiple doses can also be administered at intervals of 6-24 hror greater.

The use of tat variant peptides having reduced capacity to bind to andinhibit N-type calcium channels is particularly useful in patientshaving above normal susceptibility to side effects resulting frombinding to these channels. These include patients have normal (systolic120-129 mm Hg and diastolic 80-84 mm Hg) or below normal blood pressure.Subnormal blood pressure can arise as a result of blood losscontemporaneously with insult to the CNS (for example, in a patient whoexperiences traumatic injury in a car accident, or in a patient whoincurs blood loss as a result of a fall following stroke).

The response of the patient to the administration of a chimeric peptideor peptidomimetic of the invention can be monitored by determininginfarction volume before and at various times after treatment. Earlyischemia is detectable using MRI diffusion imaging. Combinations of MRIprotocols, including perfusion imaging, can be used to determine tissueat risk and predict infarction volume. The methods preferably achieve areduction in infarction volume of at least 10, 15, 20, 25, 30, 35, 40,or 50% relative to the mean infarction volume in a population ofcomparable patients not receiving treatment by the methods of theinvention. The response of the patient can also be measured from adisability index determined one day to one week after initiatingtreatment. The patient preferably shows an improvement (i.e., lessdisability) in disability index of at least 4, 10, 15, 20, 25, 30, 35,40, or 50% relative to the mean disability index in a population ofcomparable patients not receiving treatment by the methods of theinvention The patient preferably scores a zero or one on the Rankinstroke index or over 75 on the Barthel index.

VIII. Pharmaceutical Compositions, Dosages and Routes of Administration

The chimeric peptides or peptidomimetics of the invention can beadministered in the form of a pharmaceutical composition. Pharmaceuticalcompositions are manufactured under GMP conditions. Pharmaceuticalcompositions can be provided in unit dosage form (i.e., the dosage for asingle administration) containing any of the dosages indicated above.Pharmaceutical compositions can be manufactured by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes. In particularly,lyophilized chimeric peptides or peptidomimetics of the invention can beused in the formulations and compositions described below.

Pharmaceutical compositions can be formulated in conventional mannerusing one or more physiologically acceptable carriers, diluents,excipients or auxiliaries that facilitate processing of chimericpeptides or peptidomimetics into preparations which can be usedpharmaceutically. Proper formulation is dependent on the route ofadministration chosen.

Administration can be parenteral, intravenous, oral, subcutaneous,intraarterial, intracranial, intrathecal, intraperitoneal, topical,intranasal or intramuscular. Intravenous administration is preferred.

Pharmaceutical compositions for parenteral administration are preferablysterile and substantially isotonic. For injection, chimeric peptides canbe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological saline or acetate buffer (to reduce discomfort at the siteof injection). The solution can contain formulatory agents such assuspending, stabilizing and/or dispersing agents.

Alternatively the chimeric peptides or peptidomimetics can be in powderform for constitution with a suitable vehicle, e.g., sterilepyrogen-free water, before use.

For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. This route ofadministration can be used to deliver the compounds to the nasal cavityor for sublingual administration.

For oral administration, the chimeric peptides or peptidomimetics can beformulated with pharmaceutically acceptable carriers as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions and thelike, for oral ingestion by a patient to be treated. For oral solidformulations such as, for example, powders, capsules and tablets,suitable excipients include fillers such as sugars, such as lactose,sucrose, mannitol and sorbitol; cellulose preparations such as maizestarch, wheat starch, rice starch, potato starch, gelatin, gumtragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone (PVP); granulatingagents; and binding agents. If desired, disintegrating agents can beadded, such as the cross-linked polyvinylpyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate. If desired, solid dosageforms can be sugar-coated or enteric-coated using standard techniques.For oral liquid preparations such as, for example, suspensions, elixirsand solutions, suitable carriers, excipients or diluents include water,glycols, oils, alcohols. Additionally, flavoring agents, preservatives,coloring agents and the like can be added.

In addition to the formulations described previously, the chimericpeptides or peptidomimetics can also be formulated as a depotpreparation. Such long acting formulations can be administered byimplantation (for example subcutaneously or intramuscularly) or byintramuscular injection. Thus, for example, the compounds can beformulated with suitable polymeric or hydrophobic materials (for exampleas an emulsion in an acceptable oil) or ion exchange resins, or assparingly soluble derivatives, for example, as a sparingly soluble salt.

Alternatively, other pharmaceutical delivery systems can be employed.Liposomes and emulsions can be used to deliver chimeric peptides.Certain organic solvents such as dimethylsulfoxide also can be employed,although usually at the cost of greater toxicity. Additionally, thecompounds can be delivered using a sustained-release system, such assemipermeable matrices of solid polymers containing the therapeuticagent.

Sustained-release capsules can, depending on their chemical nature,release the chimeric peptides for a few weeks up to over 100 days.Depending on the chemical nature and the biological stability of thetherapeutic reagent, additional strategies for protein stabilization canbe employed.

As the chimeric peptides or peptidomimetics of the invention can containcharged side chains or termini, they can be included in any of theabove-described formulations as the free acids or bases or aspharmaceutically acceptable salts. Pharmaceutically acceptable salts arethose salts which substantially retain the biologic activity of the freebases and which are prepared by reaction with inorganic acids.Pharmaceutical salts tend to be more soluble in aqueous and other proticsolvents than are the corresponding free base forms.

The chimeric peptides or peptidomimetics are used in an amount effectiveto achieve the intended purpose (e.g., reduction of damage effect of thedamaging stroke and related conditions). A therapeutically effectiveamount means an amount of chimeric peptide or peptidomimetics sufficientto significantly reduce the damage resulting from stroke in a populationof patients (or animal models) treated with the chimeric peptides orpeptidomimetics of the invention relative to the damage in a controlpopulation of stroke patients (or animal models) not treated with thechimeric peptides or peptidomimetics of the invention. The amount isalso considered therapeutically effective if an individual treatedpatient achieves an outcome more favorable than the mean outcome(determined by infarction volume or disability index) in a controlpopulation of comparable patients not treated by methods of theinvention. The amount is also considered therapeutically effective if anindividual treated patient shows a disability of two or less on theRankin scale and 75 or more on the Barthel scale. A dosage is alsoconsidered therapeutically effective if a population of treated patientsshows a significantly improved (i.e., less disability) distribution ofscores on a disability scale than a comparable untreated population, seeLees et al., N Engl J Med 2006; 354:588-600. A therapeutically effectiveregime means a combination of a therapeutically effective dose and afrequency of administration needed to achieve the intended purpose asdescribed above. Usually a single administration is sufficient.

Preferred dosage ranges include 0.001 to 20 μmol chimeric peptide orpeptidomimetic per kg patient body weight, optionally 0.03 to 3 μmolchimeric peptide per kg patient body weight to μmol chimeric peptide perkg patient body weight within 6 hours of stroke. In some methods, 0.1-20μmol chimeric peptide or peptidomimetic per kg patient body weightwithin 6 hours are administered. In some methods, 0.1-10 μmol chimericpeptide or peptidomimetic per kg patient body weight is administeredwithin 6 hours, more preferably about 0.3 μmol chimeric peptide per kgpatient body weight within 6 hours. In other instances, the dosagesrange is from 0.005 to 0.5 μmol chimeric peptide or peptidomimetic perkg patient body weight. Dosage per kg body weight can be converted fromrats to humans by dividing by 6.2 to compensate for different surfacearea to mass ratios. Dosages can be converted from units of moles tograms by multiplying by the molar weight of a chimeric peptide orpeptidomimetic. Suitable dosages of chimeric peptides or peptidomimeticsfor use in humans can include 0.001 to 5 mg/kg patient body weight, ormore preferably 0.005 to 1 mg/kg patient body weight or 0.05 to 1 mg/kg,or 0.09 to 0.9 mg/kg. In absolute weight for a 75 kg patient, thesedosages translate to 0.075-375 mg, 0.375 to 75 mg or 3.75 mg to 75 mg or6.7 to 67 mg. Rounded to encompass variations in e.g., patient weight,the dosage is usually within 0.05 to 500 mg, preferably 0.1 to 100 mg,0.5 to 50 mg, or 1-20 mg.

The amount of chimeric peptide or peptidomimetics administered dependson the subject being treated, on the subject's weight, the severity ofthe affliction, the manner of administration and the judgment of theprescribing physician. The therapy can be repeated intermittently whilesymptoms detectable or even when they are not detectable. The therapycan be provided alone or in combination with other drugs.

Therapeutically effective dose of the present chimeric peptides orpeptidomimetics can provide therapeutic benefit without causingsubstantial toxicity. Toxicity of the chimeric peptides can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., by determining the LD₅₀ (the dose lethal to50% of the population) or the LD₁₀₀ (the dose lethal to 100% of thepopulation). The dose ratio between toxic and therapeutic effect is thetherapeutic index. Chimeric peptides or peptidomimetics exhibiting hightherapeutic indices are preferred (see, e.g., Fingl et al., 1975, In:The Pharmacological Basis of Therapeutics, Ch. 1, p. 1).

IX. Screening Methods

The invention further provides methods of screening otherinternalization peptides to determine whether such peptides bind and/orinhibit N-type calcium channels. Test peptides can be assessed for suchbinding or inhibiting, either alone or linked to an active agent,particularly an active peptide, sometimes known as a cargo peptide.Other internalization peptides that can be tested include antennapediaiinternalization peptide (Bonfanti, Cancer Res. 57, 1442-6 (1997)) (andvariants thereof), Tat variants, Penetratin, SynB1 and 3, Transportan,Amphipathic, gp41NLS, polyArg, and others described in the followingreferences (Temsamani, Drug Discovery Today, 9(23):1012-1019, 2004; DeCoupade, Biochem J., 390:407-418, 2005; Saalik Bioconjugate Chem. 15:1246-1253, 2004; Zhao, Medicinal Research Reviews 24(1): 1-12, 2004;Deshayes, Cellular and Molecular Life Sciences 62:1839-49, 2005) (allincorporated by reference).

X. Linkage of Tat Variants to Other Active Agents

The tat variants described above can be linked to any other active agentto promote uptake of the agent through cell membranes and/or the bloodbrain barrier. Use of a chimeric agent comprising or consisting of a tatvariant and active agent in a therapeutic method improvesbioavailability at the intended site relative to use of the active agentalone, and reduces side effects through binding to N-type calciumchannels relative to use of the active agent linked to a standard tatpeptide. The tat variants are particularly useful for active agents withan intracellular target and/or neuroactive drugs that need to cross theblood brain barrier to exert activity. Some but not all of the activeagents amenable to attachment of tat variants are peptides. Use of tatvariants is particularly useful for existing pharmaceuticals that havepoor bioavailabilty, high dosages or short half-lives.

Some guidance for selection of active agents, methods for attachmentsand use thereof is provided by the scientific and patent literaturerelating to previous tat peptides (see, e.g., U.S. Pat. No. 6,316,003and U.S. Pat. No. 5,804,604). All of the above description in relatingto chimeric peptides comprising an active peptide linked to a tatvariant for treatment of stroke and related diseases applies mutatismutandis to chimeric agents comprising a tat variant linked to an activeagent.

The table below lists the names of active agents (some of which areapproved drugs), the disorders they are useful for treating, whether thedisease is acute or chronic, the routes of administration of drugs (tothe extent established) and comments on problems with existing drugsthat may in part be overcome by the improved transport through membranesconferred by a tat variant peptide.

Chimeric agents comprising a tat variant peptide linked to an activeagent can be used at the same or lower dosage on a molar basis as theactive agent alone, and can be administered by the same route as theactive agent alone, and for treatment of the same disease(s) as theactive agent alone. The preferred methods of administration forpeptide:active conjugates disclosures within are intravenous,intraarterial, intranasal/inhalation, intramusular, intraperitoneal,sub-lingual, per-rectum, and topical (for disorders of the dermis orproximal to epithelial cells).

TABLE 5 Acute/ Route of Active Agent Disease chron admin CommentReference Phenobarbitol Epilepsy IV/oral Dependence, Motamedi & (luminalsodium) tolerance Meador (2006) issues, Curr Neurol interactions,Neurosci Rep, side effects, 6(4): 341-6. birth defects Drugs.comPrimidone Epilepsy Oral Side effects, Koristkova, et (myidone,interactions al (2006) Int J mysoline) Clin Pharmacol Ther, 44(9):438-42. Drugs.com Diazepam Anxiety IP/oral Dependence, Beard, et al(valium) side effects, (2003) Health interactions Technol Assess, 7(40):iii, ix-x, 1-111. Drugs.com Dopamine Parkinson's Cannot cross Ahlskog(2001) BBB, side Neurol Clin, effects 19(3): 579-605. Drugs.com LevodopaParkinson's Degraded Nyholm (2006) before BBB, Clin side effects,Pharmacokinet, halflife = 1.5 hrs 45(2): 109-36. USPTO.gov (patent #7160913) Apomorphine IP Short half-life Nyholm (2006) ClinPharmacokinet, 45(2): 109-36. Drugs.com Tirilazad Stroke IP Lowefficacy, Hickenbottom mesylate phase III & Grotta (Freedox) stopped(1998) Semin Neurol 18(4): 485-92. Strokecenter.org Cyclosporine ImmuneIP Peptide, 5-18 hr Kees, et al (Gengraf) suppression halflife (2006)Ther Drug Monit, 28(3): 312-20. Drugs.com Vacomycin Antibiotic IPPeptide, low de Hoog, et al uptake, 4-6 hr (2004) Clin halflifePharmacokinet, 43(7): 417-40. Drugs.com Lisinopril Hypertension IP/oralPeptide, poor Tan, et al (Prinivil) BBB (2005) Am J crossing, 12 hrHypertens, halflife 18(2): 158-64. Drugs.com Azidothymidine AntiviralOral Poor BBB Spitzenberger, (AZT, zidoridine, crossing, 05-3 hr et al(2006) J combivir) halflife, Cereb Blood hematologic Flow Metab,toxicology Oct 25, Epub ahead of print. Drugs.com PiracetamPain/epilepsy Cannot cross Loscher & BBB Potschka (2002) J Pharmacol ExpTher, 301(1): 7-14. US7,157,421) Natrecor Cardio-renal IV UnknownFeldman & Sun (BNP peptide) decompensation efficacy (2004) Heartsyndrome Fail Rev, 9(3): 203-8. Clinicaltrials.gov AVR-118 CancerSubcutaneous Unknown Clinicaltrials.gov (peptide) palliative efficacy,unknown dosage Oxytocin Mood IV/IM Interactions, Swaab, et al (peptide)disorders unknown (2005) Ageing dosage Res Rev, 4(2): 141-94. Drugs.comPravastatin MS Oral Unknown Hatanaka (Pravachol) efficacy, low (2000)Clin bioavailability Pharmacokinet, 39(6): 397-412. Clinicaltrials.govRemifentanil Pain, burn IV 3.5 min Scott & Perry halflife, (2005) Drugs,metabolized 65(13): 1793-1823. by unknown Clinicaltrials.gov esteraseNeurotensin Schizphrenia, 13AA Boules, et al, parkinson's, peptide,(2006) addiction easily Peptides, degraded, 27(10): 2523-33. cannotcross BBB GDNF (glial Parkinson's Chronic Intra- Peptide, Grondin, et alderived parenchymal Cannot cross (2003) Prog neurotrophic BBB Drug Res,61: factor) 101-23. Protease HIV Oral All HIV Oldfield & inhibitorsprotease Plosker (2006) lopinavir inhibitors Drugs 66(9): ritonavirsuffer from 1275-99. saquinavir the acute Porter & darunavir capacity ofCharman atazanavir HIV to (2001) Adv amprenavir mutate, Drug Delivgenerating Rev, Oct 1; 50 drug resistant Suppl 1: S127-47. HIV strainsPiacenti (2006) Pharmacotherapy 26(8): 1111-33. DihydroergotamineMigraine IV, IM, sub-Q Interactions Modi & cause Lowder (2006)peripheral Am Fam ischemia, 9 hr Physician halflife 73(1): 72-8.Sporamax Antifungal Oral Drug Wang & (itaconazole) resistance Remold(2006) eventually Cardiol Rev develops, 14(5): 223-6. congestive heartfailure in some populations Protein Kinase C Acute US pat inhibitorsmyocardial publications infarction, 20050267030, stroke, 20060148702,ischemia, 20060293237, reperfusion 20050215483, injury 20040204364,20040009922 AII-7 Cancer, breast Chronic Peptidomimetic Kunz et al, Molcancer that blocks Cancer Res Erbb2 2006; 4(12): 983-98 intracellulardomain and increases taxol sensitivity CRAMP peptide SalmonellaIntracellular Rosenberger, infection anti-microbial CM. PNAS| peptidethat Feb. 24, reduces 2004|vol. 101| Salmonella no. 8|2422-2427replication Sodium channel May reduce Peptide Vassilev, peptide musclecorresponding Science (1988) spasms to the short 241: 1658-6 (epilepsy,intracellular restless leg, segment parkinsons, between etc) homologoustransmembrane domains III and IV of sodium channel alpha subunit slowedinactivation Aptamer KDI1 Blocks EGF Buerger. J. signaling — Biol.Chem., possible anti Vol. 278, Issue cancer 39, 37610-37621, Sep. 26,2003 RNA/gene Transporter Turner et al therapy peptides can (2007) Bloodbe used to Cells Mol Dis, bring in 38(1): 1-7. RNAs or siRNA/RNAi fortreatment

Example 1 Screening for Side Effects of Tat-NR2B9c

Tat-NR2B9c is a chimeric peptide of a standard tat peptide and KLSSIESDV(SEQ ID NO:12) previously shown to be effective in a rat model ofstroke. This example screens the peptide Tat-NR2B9c for capacity toinhibit binding of known ligands to about 70 receptors proteins.Examples of receptors screened included glutamate, histamine H1,potassium channels, Dopamine D1, calcium channels (L-type, N-type).

Tat-NR2B9c was found to inhibit binding to two such receptors, an N-typecalcium channel and a chemokine CXCR2 (IL-8Rb). The screen was performedas a competitive binding assay in which unlabelled Tat-NR2B9c at aconcentration of 10 μM competed with an I125 labeled ligand for bindingto its receptor in the presence of unlabeled ligand to increasesensitivity. At 10 μM, Tat-NR2B9c showed 100% inhibition of radiolabeledω-Conotoxin GVIA binding to N-type Ca channels. Tat-NR2B9c also showed80% inhibition of IL-8/IL-8RB at the same concentration. Results areshown in FIGS. 1A, B, C.

Example 2 Mutagenesis of a Standard Tat Peptide

Like the known N-type calcium channel inhibiter Ziconotide, Tat-NR2B9ccontains numerous positive charges. The positive charges presumablyfacilitate both the ability to cross the blood brain barrier and mayalso contribute to N-type calcium channel binding. Direct sequencecomparison shows some similarity in positive (R=Arginine, K=Lysine)charges as well as spacing of these charges along the peptide backbone(see alignment below). This approximately maps the Tat-NR2B9c N-typecalcium channel binding epitope to the Tat region (shown in italics) andone amino acid of the NMDAR2B domain.

(SEQ ID NO:17) Y GRKKRRQRRR KLSSIESDV (Tat-NR2B9c) (SEQ ID NO:72)CKGKGAKCSRLMYDCCTGSCRSGKCG (Ziconotide)

The present inventors hypothesized that mutation of the Y residue atposition 1 of Tat-NR2B9c to F might reduce binding to N-type calciumchannels without impairing cellular uptake of the drug. The inventorsalso hypothesized that modifications of a stretch of basic residues inthe standard tat peptide would achieve a similar result. The peptideswere each applied at 100 μM. The following peptides were tested (theCa²⁺ current in shown as a percentage after each peptide): 1990 TAT:YGRKKRRQRRR (SEQ ID NO:1) (57+/−1.6% (n=5)); 1991 2B9c: KLSSIESDV (SEQID NO:12) (94+/−1.7% (n=5)); 1992 Tat-NR2B9c-AA; YGRKKRRQRRRKLSSIEADA(SEQ ID NO:18) (74+/−2.4% (n=6)); 1993 F-Tat-NR2B9c:FGRKKRRQRRRKLSSIESDV (SEQ ID NO:19) (91+/−1.6% (n=5)); 1994 Tat-NR2B9c Kto A: YGRKKRRQRRRALSSIESDV (SEQ ID NO:20) (77+/−1.8% (n=7)); 1995F-Tat-NR2B9c K to A: FGRKKRRQRRRALSSIESDV (SEQ ID NO:21) (97+/−0.2%(n=6)); 1976: YGRKKRRQRRRKLSSIESDX (SEQ ID NO:22) where X=norvaline(66+/−3.4% (n=6)); 1977: YGRKKRRQRRRKLSSIESDX (SEQ ID NO:23) whereX=L-t-butyl-glycine (65+/−5.1% (n=5)); 1987: D-isomer of Tat-NR2B9c(82+/−2.2% (n=6)). Tat-NR2B9c (68+/−1.7% (n=7)). Data were plotted asmean+/−s.e.m.

The peptides were also tested in the following patch clamp assay.Internalization peptides and chimeric peptides were screened for theircapacity to inhibit ionic currents mediated by N-type calcium channels.This was carried out by performing whole-cell patch clamp recordings indorsal root ganglion neurons, in which N-type calcium currents areexpressed. Cultures of dorsal root ganglions (DRGs) were prepared fromSwiss mice at 13-14 d of gestation. In brief, DRG's were dissected andsubjected to trypsin digestion for 20 min at 37° C., mechanicallydissociated and plated on cover slips coated with poly-D-lysine. Theywere grown in serum free MEM (Neurobasal MEM, B-27—Gibco InvitrogenCorporation, Carlsbad, Calif.). After 3-5 days, 10 μM FUDR solution wasadded to inhibit glial proliferation. The cultures were maintained at37° C. in a humidified 5% CO₂ atmosphere and were fed twice a week.Whole-cell recording were carried out at room temperature 10-14 daysafter plating. Electrophysiology recordings: Whole-cell recordings wereperformed with an Axopatch-1B amplifier (Axon Instruments, Foster City,Calif.) in the voltage-clamp mode. Recording electrodes, withresistances of 3-5 MΩ, were constructed from thin-walled borosilicateglass (1.5 mm diameter; World Precision Instruments, Sarasota, Fla.)using a two-stage puller (PP83; Narishige, Tokyo, Japan). Data weredigitized, filtered (2 kHz), and acquired on-line using the programs ofpClamp 9 (Axon Instruments). The pipettes were filled with a solutioncontaining (mM): CsCl 110, MgCl2 3, EGTA 10, HEPES10, MgATP 3, GTP 0.6.The pH was adjusted to 7.2 with CsOH. The bath solution contained (mM):CaCl2 1, BaCl2 10, HEPES10, TEA-Cl 160, Glucose 10, TTX 0.0002 at pH(NaOH) 7.4. Whole-cell currents were elicited using 40 ms depolarizingpulses to +20 mV from a holding potential of −60 mV, applied every 15 s.To test the use-dependent inhibition, currents were elicited using 10 msdepolarizing pulses to +20 mV from a holding potential of −60 mV,applied every 0.02 s (50 Hz), 0.05 s (20 Hz), 0.1 s (10 Hz) or 15 s(0.07 Hz) respectively.

Results: The results are presented in FIG. 2. The upper portionrepresents the means+/−s.e.m. of whole cell calcium current in thepresence of the indicated peptide normalized to the whole cell calciumcurrent in the same cells before application of the peptide. The lowerportion of FIG. 2 shows representative whole-cell traces from which themeans in the upper portion were derived. In brief, the data show thatthe TAT transporter portion of the chimeric peptide is predominantlyresponsible for the inhibition of N-type calcium channels. Mutation ofthe N-terminal tyrosine of Tat-NR2B9c almost completely abrogates theability of this chimeric peptide to inhibit N-type calcium channels. TheC-terminal portion of Tat-NR2B9c (KLSSIESDV (SEQ ID NO:12)),F-Tat-NR2B9c or 1994 Tat-NR2B9c K to A showed no significant inhibitionof N-type calcium channel activity. Peptides 1992, 1994 and 1987 showedsignificant improvement in channel activity over TAT alone althoughstill displayed some reduction in the amount of N-type calcium channelactivity. All of these peptides provide reduced binding to N-typecalcium channels over standard Tat alone that indicate an increasedtherapeutic index of a drug that includes one of these Tat variantsequence.

Example 3 Further Analysis of Inhibition of N-Type Ca²⁺ Channel-MediatedIonic Currents by Tat-NR2B9c

Further experiments were carried out as depicted in FIGS. 4-7. Theirpurpose was to further characterize the inhibition of N-type Ca²⁺channel-mediated ionic currents by Tat-NR2B9c. Additionally, FIG. 4characterizes the degree of inhibition of the Ca²⁺ current by Tat-NR2B9c(YGRKKRRQRRRKLSSIESDV, SEQ ID 17) and this is compared with the othervariants: 1990 TAT (YGRKKRRQRRR, SEQ ID NO:1); 1992 Tat-NR2B9c AA(YGRKKRRQRRRKLSSIEADA, SEQ ID 18); 1994 Tat-NR2B9c KtoA(YGRKKRRQRRRALSSIESDV, SEQ ID 20); 1987 D-Tat-NR2B9c(YGRKKRRQRRRKLSSIESDV (all D-amino acids), SEQ ID); 1976(YGRKKRRQRRRKLSSIESDX, where X=norvaline, SEQ ID NO:22); 1977(YGRKKRRQRRRKLSSIESDX, where X=L-t-butyl Glycine, SEQ ID NO:23).

Tissue culture: Cultures of dorsal root ganglions (DRGs) were preparedfrom Swiss mice at 13-14 d of gestation. Briefly, DRG's were dissectedand subjected to trypsin digestion for 20 min at 37° C., mechanicallydissociated and plated on cover slips coated with poly-D-lysine. Theywere grown in serum free MEM (Neurobasal MEM, B-27—Gibco InvitrogenCorporation, Carlsbad, Calif.). After 3-5 days, 10 μM FUDR solution wasadded to inhibit glial proliferation. The cultures were maintained at37° C. in a humidified 5% CO₂ atmosphere and were fed twice a week.Whole-cell recording were carried out at room temperature 10-14 daysafter plating.

Electrophysiology recordings: Whole-cell recordings were performed withan Axopatch-1B amplifier (Axon Instruments, Foster City, Calif.) in thevoltage-clamp mode. Recording electrodes, with resistances of 3-5 MΩ,were constructed from thin-walled borosilicate glass (1.5 mm diameter;World Precision Instruments, Sarasota, Fla.) using a two-stage puller(PP83; Narishige, Tokyo, Japan). Data were digitized, filtered (2 kHz),and acquired on-line using the programs of pClamp 9 (Axon Instruments).The pipettes were filled with a solution containing (mM): CsCl 110,MgCl2 3, EGTA 10, HEPES 10, MgATP 3, GTP 0.6. The pH was adjusted to 7.2with CsOH. The bath solution contained (mM): CaCl2 1, BaCl2 10, HEPES10, TEA-Cl 160, Glucose 10, TTX 0.0002 at pH (NaOH) 7.4. Whole-cellcurrents were elicited using 40 ms depolarizing pulses to +20 mV from aholding potential of −60 mV, applied every 15 s. To test theuse-dependent inhibition, currents were elicited using 10 msdepolarizing pulses to +20 mV from a holding potential of −60 mV,applied every 0.02 s (50 Hz), 0.05 s (20 Hz), 0.1 s (10 Hz) or 15 s(0.07 Hz) respectively. Data analysis: Data were plotted asmean+/−s.e.m.

FIG. 4 demonstrates that increasing concentrations of all peptidescontaining an intact Tat sequence (YGRKKRRQRRR (SEQ ID NO:1))significantly inhibit Ca²⁺ currents in dorsal root ganglion neurons(which express predominantly N-type Ca²⁺ channels). This suggests thatthe property of inhibiting N-type Ca²⁺ channel currents resides in theTat sequence.

FIGS. 5A and B demonstrate that the inhibition of the Ca²⁺ current byTat-NR2B9c is specific to N-type Ca²⁺ channels. Omega conotoxin (1 μM),a selective N-type Ca²⁺ channel blocker, inhibits the Ca²⁺ current, andno additional inhibition is afforded by Tat-NR2B9c (100 μM) onceN-channels are blocked (FIG. 5A, left). Similarly, no additionalinhibition of the current is seen when conotoxin is added after theinhibition of the ionic current by Tat-NR2B9c (FIG. 5A, right). Also,the selective L-type Ca²⁺ channel blocker, nifedipine, doessignificantly affect the size of the Ca²⁺ current recorded in thepresence (100 μM intracellular), or absence of, Tat-NR2B9c as shown inFIG. 5B. The left portion of FIG. 5B shows the means+/−s.e.m.s ofcalcium currents, whereas on the right are representative traces ofwhole cell currents from a single experiment.

FIG. 6 demonstrates that the block of Ca²⁺ currents by Tat-NR2B9c is notfrequency dependent. 100 μM Tat-NR2B9c was used to test itsuse-dependent effect. The currents elicited by depolarizing pulses of+20 mV showed strong frequency-dependent rundown. However, the increaseof frequency (0.07, 10, 20, 50 Hz) did not increase Tat-NR2B9c'sinhibition effect on this current. The figure shows Ca²⁺ currentsrecorded in one representative DRG neuron at different frequencies.These currents have a natural tendency to run-down after a few minutes,and the increase in frequency had no effect on the inhibition of thecurrent by Tat-NR2B9c (representative of n=4).

FIG. 7 demonstrates that Tat-NR2B9c inhibits the Ca²⁺ current in DRGneurons in a manner that is independent of voltage, and that thisinhibition is specific to N-type Ca²⁺ channels because it is notaffected by nifedipine, a blocker of L-type Ca²⁺ channels. The currentswere elicited using 50 ms voltage-clamp steps from −40 to +50 mV fromthe holding potential of −60 mV.

In conclusion, FIGS. 4-7 show that the inhibition of Ca²⁺ currents byTat-NR2B92 is specific to N-type Ca²⁺ channels, and is similarly aproperty of other peptides bearing the Tat moiety. The data also showthat this inhibition is specific to N-type Ca²⁺ channels, and isindependent of frequency and of voltage.

Example 3 F-Tat-NR2B9c is Equally Effective in a Stroke Model

F-Tat-NR2B9c was compared with Tat-NR2B9c at a single does of 3 mmol/gweight in the rat pial occlusion model of permanent ischemia describedabove and further in example 4. In both cases, the chimeric peptide wasadministered one hour after initiating ischemia. F-Tat-NR2B9c andTat-NR2B9c were equally effective in reducing infarct size as shown inFIG. 3.

Example 4 Purpose

-   1. To test the neuroprotective efficacy of the Tat-NR2B9c peptide in    both male and female rats using the in vivo pial 3 vessel occlusion    (P3VO) model of stroke.-   2. To elucidate the mechanism of action by testing 6 variations of    the Tat-NR2B9c peptide in male rats.

Background

The peptide known as Tat-NR2B9c has been developed and previously testedin the MCAO model of stroke in the rat. This peptide has been shown tobe neuroprotective as seen by a reduced infarct size. However, the MCAOmodel of stroke results in a large infarct with extensive neurologicaldeficits and shortened life span. The P3VO model of stroke results in amuch smaller, cortical infarct with minimal neurological deficit andnormal life span.

Six additional peptides were tested that contain the same amino acidsequence as Tat-NR2B9c except for the terminal 3 amino acids. By varyingthese amino acids and then testing the neuroprotective efficacy of thepeptides in the P3VO model of stroke, the mechanism of action can befurther elucidated.

The amino acid structure of Tat-NR2B9c and the 6 peptides are asfollows:

Sequence: Name: YGRKKRRQRRRKLSSIESDV Tat-NR2B9c (SEQ ID NO:17)YGRKKRRQRRRKLSSIESDX X = 3-fluoro-DL-Valine 1974 (SEQ ID NO:73)YGRKKRRQRRRKLSSIETDX X = norvaline 1975 (SEQ ID NO:74)YGRKKRRQRRRKLSSIESDX X = norvaline 1976 (SEQ ID NO:22)YGRKKRRQRRRKLSSIESDX X = L-t-butyl-glycine 1977 (SEQ ID NO:23)YGRKKRRQRRRKLSSIEXDV X = L-2-amino-3-ureidopropionic acid 1978 (SEQ IDNO:75) YGRKKRRQRRRKLSSIETAL 1980 (SEQ ID NO:76)

Methods

Animals

Adult Sprague Dawley rats (10-12 weeks old) (males˜300 g, females˜250 g)(FIG. 8) were fasted for 12-18 hours before being subjected to permanentpial vessel occlusion of 3 terminal branches of the Middle CerebralArtery over the Whisker Barrel Cortex (P3VO). Each of 7 peptides weretested in male rats plus a saline control group (n=8 in each group). TheTat-NR2B9c peptide and a saline control group were tested in female rats(n=8 in each group). The researcher was blinded to the treatment groupduring the time of surgery through to the analysis of infarct size.

General Procedure

Rats were anesthetized with a 0.5 ml/kg intramuscular injection ofketamine (100 mg/kg), acepromazine (2 mg/kg), and xylazine (5 mg/kg),supplemented with one third the initial dose as required. An analtemperature probe was inserted and the animal was placed on a heatingpad maintained at 37° C. The right external carotid artery (ECA) wascannulated with PE 10 polyethylene tubing for dye injections. The skullwas exposed via a midline incision, scraped free of tissue, and thetemporalis muscle disconnected from the skull on the right side. Using adissecting microscope and a pneumatic dental drill, a 6×4 mm cranialwindow was made over the right somatosensory cortex (2 mm caudal and 5mm lateral to bregma) by drilling a rectangle through the skull andlifting off the piece of skull while keeping the dura intact. Afterbeing bathed with artificial cerebrospinal fluid, small boluses (10 to20 μL) of the vital dye patent blue violet (10 mmol/L; Sigma) in normalsaline, were injected into the right external carotid artery todemonstrate transit through surface vessels of the cortex. Threecritical arteriolar branches of the MCA around the barrel cortex wereselected and electrically cauterized through the dura. After thecauterizations, the bolus injections and dye transits were repeated toensure transits through the cauterized arterioles were blocked. Therectangle of skull was replaced over the window and the scalp wassutured. The catheter was removed from the ECA, the ECA was ligated, andthe anterior neck was sutured. One hour after initiation of focalocclusion, 0.3 ml of drug (3 nMol/g body weight) or saline control wereinfused through the tail vein at a rate of 0.06 ml/min. Each rat wasreturned to its individual cage under a heating lamp to maintain bodytemperature until the rat fully recovered. Food and water was suppliedad libitum.

Harvesting of Brain Tissue

Twenty-four hours post-surgery, animals were re-anesthetized with 1 mLpentobarbital and the brain was quickly harvested. One coronal slice wastaken through the infarct region and incubated in 2%triphenyltetrazolium chloride (TTC) for 15 minutes at 37° C. Images werescanned and brain slices were stored at −80° C.

Analysis

Infarct size was measured as a percent of the hemisphere for each rat inthe study. After obtaining infarct size measurements, the animals wereseparated into their respective groups. Comparisons were made betweentreatment groups as means±SE.

Results

Of the six novel peptides tested, Tat-NR2B9c, 1976 and 1977 resulted ina significantly decreased infarct sizes and 1975 and 1978 displayed somereduction in infarct size (FIG. 8).

All publications, and patent filings cited in this specification areherein incorporated by reference as if each individual publication orpatent were specifically and individually indicated to be incorporatedby reference. Genbank records referenced by Genbank identification (GID)or accession number, particularly any polypeptide sequence,polynucleotide sequences or annotation thereof, are incorporated byreference herein. If more than one version of a sequence has beenassociated with the same accession number at different times, referenceto a deposit number should be construed as applying to the version inexistence at the effective filing date of the application dating back toa priority application if the deposit is also referenced in the priorityapplication. Various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. Unless otherwise apparent from the context, any feature, stepor embodiment can be used in combination with any other feature, step orembodiment.

1. An isolated chimeric peptide, wherein the chimeric peptide comprisesan active peptide that inhibits binding of PSD-95 to an NMDA receptor,the active peptide having an amino acid sequence comprising ESDV (SEQ IDNO:6) or ETDV (SEQ ID NO:8) and an internalization peptide that promotesuptake of the chimeric peptide into cells and has reduced capacity tobind to an N-type calcium channel relative to the tat peptideYGRKKRRQRRR (SEQ ID NO:1), wherein the internalization peptide is a tatpeptide that is a variant of SEQ ID NO:1 in which the N-terminal Y issubstituted with F.
 2. The isolated chimeric peptide of claim 1, whereinthe active peptide has an amino acid sequence comprising KLSSIESDV (SEQID NO:12).
 3. The isolated chimeric peptide of claim 1, wherein theactive peptide has an amino acid sequence comprising KLSSIETDV (SEQ IDNO:13).
 4. The isolated chimeric peptide of claim 1, wherein thechimeric peptide has an amino acid sequence comprisingFGYKKRRQRRRKLSSIESDV (SEQ ID NO:14) or FGYKKRRQRRRKLSSIETDV (SEQ IDNO:16).
 5. The isolated chimeric peptide of claim 1, wherein thechimeric peptide has an amino acid sequence consisting ofFGYKKRRQRRRKLSSIESDV (SEQ ID NO:14) or FGYKKRRQRRRKLSSIETDV (SEQ IDNO:16).
 6. The isolated chimeric peptide of claim 1, wherein thechimeric peptide has a Kd greater than 10 nM for an N-type calciumchannel.
 7. A composition comprising the isolated chimeric peptide ofclaim 1 and a pharmaceutically acceptable carrier.